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Carbohydrate-deficient transferrin (CDT) is the most specific marker for diagnosis of chronic excessive alcohol consumption and includes the serum transferrin (Tf) isoforms with two or less sialic acid residues (di-, mono-, and asialo-Tf). To monitor serum CDT, we developed a capillary zone electrophoresis (CZE) method based on the dynamic capillary coating with diethylenetriamine (DETA). The separation was performed in a bare fused-silica capillary (50 microm ID, 57 cm in length), applying a voltage of 25 kV and a temperature of 40 degrees C. Using a 100 mmol/L borate buffer, pH 8.4 with 3 mmol/L DETA, the Tf isoforms (asialo- to pentasialo-Tf) were resolved within 16 min. Enzymatic cleavage of sialic acid residues with neuraminidase and immunosubtraction were used to identify CDT isoforms. The relative amount of CDT expressed as area % of disialo-Tf isoform related to the area of tetrasialo-Tf in 50 healthy donors (24 males and 26 females; aged 25-50 years) was 3.15 +/- 0.76% (mean +/- SD). The comparison between CDT values obtained by this CZE procedure and the "Axis-Shield %CDT" kit gave r = 0.644, p < 0.001 (n = 290). This easy to use and inexpensive CZE procedure could be an ideal tool to investigate CDT proteins for clinical or forensic purposes.

Transferrin sialoforms with fewer than three sialic acid residues (carbohydrate deficient transferrin; CDT) have been implicated as a marker of certain liver pathologies. Transferrin sialoforms in human sera from alcoholic and non-alcoholic patients was analyzed by capillary electrophoresis (CE) using diaminobutane (DAB) to dynamically-coat the capillary wall to minimize protein-wall interactions. Using a DAB concentration of 3 mM, transferrin sialoforms were adequately resolved to allow for direct detection of CDT without extensive treatment of the sera. Serum immunoglobulins, which migrated close to the CDT region, were removed via subtraction with protein A, enhancing the detection of CDT. The reproducibility of sialoform separation in dynamically-coated capillaries was found to be acceptable with run-to-run relative standard deviation values of 0.15% for a sample on a given day and 0.29+/-0.06% for four samples day-to-day. These results suggest that dynamic-coating approaches may provide a simple alternative to the use of covalently-coated capillaries for the CE separation of complex samples.

Abnormal agar gel electrophoretic patterns of alkaline phosphatase (AP) isoenzymes have been demonstrated in 11 out of 16 chronic alcoholics in spite of normal or borderline routine liver tests (total AP, GOT, GPT, bilirubin). The abnormality consisted of the presence of a fast-moving α1-fraction known to be related to hepatic disease preferably of obstructive type. The presence of α1-AP appears to be a more sensitive indicator of ethanol-induced liver damage than LDH-5 and OCT.

The new biochemical marker of chronic alcohol abuse are transferrin isoforms with a reduced number of sialic acids (asialo-, monosialo-, and disialotransferrin) called carbohydrate-deficient transferrins (CDTs). The usefulness of this indicator in the diagnostics of alcohol abuse has clearly been confirmed during the last years. The sensitivity and specificity of the CDT method as a marker of chronic alcohol abuse are higher than those found in commonly used tests. In routine analysis, CDTs have been assayed in the laboratories for ten years. Current CDT techniques allow for the determination of total CDT concentration (the absolute and/or relative amount) and provide the possibility of assaying its isoforms. In this paper the molecular structure of transferrin and different methods of CDT analysis are shown, with particular attention given to commercial tests. Analytical specificity and method-dependent (electrophoretic and chromatographic) reference values or cut-offs for serum CDT concentrations indicating chronic alcohol abuse are given in a table. Electrophoretic methods are characterized by higher selectivity (genetic variants) and higher sensitivity than chromatographic techniques. Isoelectric focusing, capillary electrophoresis, and high-performance liquid chromatography are used as the reference methods. Commercial procedures are based on transferrin saturation with iron followed by fractionation of protein (separating CDTs from non-CDT forms) by ion-exchange chromatography using microcolumns and a quantitative determination of CDT isoforms by immunological methods (radioimmunoassay, enzyme immunoassay, and turbidimetric immunoassay). Due to the different existing analytical methods, further standardization of CDT analysis and a redefinition of CDTs are necessary.

Carbohydrate-deficient transferrin (CDT) is currently considered to be the best available marker for the diagnosis of chronic alcoholism. A large variety of methods have been developed, demonstrating the need for standardisation. Commercially available anion-exchange chromatographic-based assays are easy to use and require no specialised, expensive instruments. However, these methods cannot identify genetic transferrin variants or the carbohydrate-deficient glycoprotein syndrome. In 1989, a capillary isoelectric focusing method was developed for quantitative measurement of CDT. Despite the optimal resolution, this method is not easily applied in a clinical routine environment due to the complexity of analysis. Capillary electrophoresis in a polymer network using coated capillaries allowed full resolution of the sialoforms of human transferrin. The drawbacks due to an expensive and time-consuming sample preparation were eliminated when a method in neat serum was developed. Capillary zone electrophoresis allowed full resolution of the transferrin isoforms with a high analytical performance in a short analysis time thanks to a strong electroosmotic flow. Genetic transferrin variants were easily detected, avoiding false-positive results. Also, using capillary zone electrophoresis, it was shown that CDT is a suitable marker of chronic alcohol abuse detection in transferrin CD (common/cathodal) variants.

Carbohydrate deficient transferrin (CDT) is one of the most reliable markers of chronic alcohol abuse. It consists of a group of minor isoforms of human transferrin (the main iron transport serum protein) deficient in sialic acid groups (asialo, monosialo and disialo) with a pI > 5.7, while the main isotransferrin (tetrasialo) has a pI of 5.4. The aim of the present work was to develop a capillary electrophoretic method to determine CDT in serum, suitable for routine use as a confirmatory technique of the current screening methods based on immunoassays. Serum samples (0.5 mL) were saturated with iron by incubation with 10 mM FeCl3 (9 microL) and 500 mM NaHCO3 (12 microL) for 30 min, then diluted 1/10 in water and injected by positive pressure (0.5 psi for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (20 microm ID, 37 cm in length) and a buffer composed of 100 mM sodium tetraborate adjusted with boric acid to pH 8.3. Applied voltage was 10 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were separated in human serum. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-transferrin, and 0.4% of trisialo-transferrin, expressed as percentages of the terasialo-transferrin peak area. Relative standard deviations (RSD) of absolute migration times were < 1%, while RSD of relative migration times (on the basis of tetrasialo-transferrin) were < 0.1%. Intra-day and day-to-day peak quantitation precision studies showed RDS ranging from 4 to 9% and from 13 to 24% for disialo- and trisialo-transferrin, respectively. The results from 30 control subjects, including social drinkers, and 13 alcoholics showed disialo- and trisialo-transferrin significantly increased in patients by a factor of about 4.5 (P < 0.0001).

To examine the dopamine D2 receptor (DRD2) gene coding sequences for abnormalities associated with schizophrenia or alcoholism and thereby help to resolve the controversy surrounding the reported association of alcoholism with a restriction fragment length polymorphism located close to the DRD2 gene.Mutational analysis of complete DRD2 gene coding sequences by denaturing gradient gel electrophoresis followed by direct nucleotide sequencing of detected variants.Patients and controls from clinical and epidemiologic collections in the United States and Europe.A total of 253 unrelated individuals, including 106 patients with schizophrenia, 113 with alcoholism, and 34 controls. For alcoholism we included patients from previously published series in which an association of illness with allele A1 was reported (Taql site 3' to the DRD2 gene) and from other published series in which nonconfirmations of this association were reported. Nearly all persons examined were white.Frequency of nonsilent variations in DRD2 gene DNA sequences in the different diagnostic groups.We found three infrequent DNA variants that predict altered amino acid sequence of the receptor. None of these is associated with either alcoholism or schizophrenia.No structural coding abnormalities in the DRD2 gene are present in alcoholism or schizophrenia.

Several analytic techniques are used to estimate the activity of creatine kinase (CK) isoenzymes. These techniques are undergoing continual refinement because the MB CK molecule appears to be nearly cardiospecific. An improved column chromatographic assay is presented with a lower limit of analytic detection of 0.8 mlU/ml. The assay was applied to normal subjects and to patients with myocardial infarction and with complex disease states. Results of the column chromatography were compared with those of agarose and cellulose acetate electrophoresis. There was MB CK activity in all normal sera (no. = 19, mean +/- standard deviation = 2.4 +/- 7). After acute myocardial infarction the MB CK activity increased by 650 percent (no. = 86, mean +/- standard error of the mean = 158.2 +/- 12.6 mlU/ml). The sensitivity for myocardial infarction was 100 percent. The specificity, evaluated in multiple clinical states, approached 100 percent. The column assay was compared with both agarose and cellulose acetate electrophoresis, and poor correlations with these semiquantitative systems were demonstrated. Finally, peak MB CK activity was closely correlated with kinetic analyses used to estimate infarct size (r = 0.94). Thus, column chromatography is a sensitive and specific method of estimating MB creatine kinase activity and is more precise than current electrophoretic methods.

HIGH concentrations of particulate triglycerides impart a characteristic milky turbidity to the blood serum known as lactescence.1 Lactescence of the serum in acute pancreatitis, supposedly a rare manifestation of this disorder, was first reported in 1865.2 In more recent publications, however, hyperlipemia was noted in 4 to 8 per cent,3 4 5 and in a report from this institution,6 the incidence of serum lipid aberrations during the course of acute pancreatitis was found to be 53 per cent. Although different mechanisms have been offered to explain the transient hyperlipemia accompanying acute pancreatitis in man and laboratory animals7 the relation between pancreatitis and . . .

A new electrophoresis of lipoproteins was examined as a means of defining hyperlipoproteinemia phenotypes. The serum lipoproteins of 119 patients with hyperlipoproteinemia whose serum total cholesterol (TC) concentration was more than 250 mg and/or whose triglyceride (TG) concentration exceeded 180 mg per 100 ml were determined by using a method of electrophoretic separation of lipoproteins on agarose and enzymic staining of each lipid component separately. Based on the lipoprotein-lipid profile with the acid of various parameters for distinction, i.e., Type II A, LDL-C greater than or equal to 180 mg/dl: Type IV, VLDL-TG greater than or equal to 130 mg/dl: type II B, LDL-C greater than or equal to 130 mg/dl, 119 patients were classified into Fredrickson's phenotypes of hyperlipoproteinemia. The percentages of phenotypes with those hyperlipoproteinemias were: type II A, 31.1%; II B, 33.6; IV, 29.4%; and V, 5.9% respectively. But types I and III were not observed in this survey. The characteristics of the lipoprotein profiles of each phenotype were clearly identified, and they were: type II A, high peak LDL-C and decrease of HDL-C fraction; II B, high peak of LDL-C and VLDL-TG; IV, high peak of VLDL-TG; V, prominence of chylomicrons and high VLDL-TG peak. The serial observations of the lipoprotein-lipid profiles were very useful for detecting the progressive changes in lipoproteins which occurred during the course of diseases, particularly in cases of secondary hyperlipoproteinemia. In conclusion, the new lipoprotein-lipid profile based on the agarose electrophoresis-enzymic staining method not only offers great help in classification of hyperlipoproteinemia but has contributed to therapeutic management.

Incubation of acetaldehyde with hemoglobin has been shown by chromatographic methods to result in an increase in fast hemoglobin. We have studied this increase in fast hemoglobin concentration by a gel electrophoretic method. Increases in fast hemoglobin concentration are detected following incubation with concentrations of 100 microM acetaldehyde and no further increase in yield occurs with concentrations greater than 7 mM acetaldehyde. The reaction is complete within 5 hr and the product is stable with a half-life of greater than 12 days.

Serial studies of plasma lipids and lipoproteins were performed in 4 patients with acute alcoholic liver disease characterized by a massive fatty liver and laboratory evidence of intrahepatic cholestasis. There were striking alterations in the plasma lipoprotein electrophoretic patterns characterized by the absence of alpha- and pre-beta-lipoprotein bands and the presence of a single band of abnormal mobility. These changes were associated with an extreme decrease in plasma lecithin-cholesterol acyltransferase activity, resulting in greatly reduced levels of plasma cholesteryl esters and increased levels of unesterified cholesterol. In 2 patients hypertriglyceridemia and hypercholesterolemia were present, the latter because of an increase in unesterified cholesterol. Lipoproteins were isolated from the plasma by sequential ultracentrifugation at the densities used for separation of normal very low density, low density, and high density lipoproteins; however, the patients' lipoproteins were different from normal in lipid composition and ultrastructure. All of the lipoprotein fractions were decreased in cholesteryl esters and the major lipoprotein was a triglyceride-rich low density lipoprotein. Electron microscopic studies of the low and high density lipoprotein fractions revealed the presence of bilamellar vesicles and stacked discs. All of the changes in lipoprotein composition and ultrastructure gradually returned to normal with clinical improvement. These observations indicate that alcoholic liver injury is associated with profound alterations in lipoprotein composition and metabolism which may be related in part to lecithin-cholesterol acyltransferase deficiency.

With a specific and sensitive electrophoretic-fluorometric method, thiamin was found in the cerebro-spinal fluid of different mammals both in free and phosphorylated form, monophosphate being the only thiamin phosphoric ester. In humans, its amount was about 60% of total thiamin. Alcoholism greatly lowered total thiamin content, affecting both thiamin forms.

Previous work in this laboratory has identified electrophoretic variant forms of arylsulfatase A in leucocyte plus platelets. During a study to replicate and extend these findings, a new seven-band variant of arylsulfatase A has been identified. Purified platelets gave a clearer, more distinct electrophoretic banding pattern than the leucocyte and platelet preparations.

A new method is presented for the determination of serum γ-glutamyl transpeptidase fractions, separated by electrophoresis on Cellogel. γ-Glutamyl-β-naphthylamide is used as the substrate and the β-naphthylamine liberated is determined by diazotization with Fast Blue B salt. Seven zones of γ-glutamyl transpeptidase activity are distinguished and classified according to their localisation in relation to the separated serum protein fractions. Studies of γ-glutamyl transpeptidase heterogeneity may be of clinical significance in the differential diagnosis of hepatic or extrahepatic diseases.

The half-lives of a number of serum enzymes were derived in a patient with acute muscle necrosis. A unit of plasma was withdrawn from the patient during the height of his illness and was i...

61 coded sera from patients with primary biliary cirrhosis (24) and various other hepatic and nonhepatic diseases (37) were tested for hepatitis B antigen (H.B. Ag.) by electron microscopy and radioimmunoassay. Investigators were not aware of the diagnosis of any patient. Particles, morphologically indistinguishable from H.B. Ag., were observed in 40 sera, including 23 which were negative for H.B. Ag. by radioimmunoassay. Assuming that radioimmunoassay is both a sensitive and reliable method for detecting H.B. Ag., there were 23 false positives and 17 true positives by electron microscopy. Of 24 patients with primary biliary cirrhosis, 5 were positive for H.B. Ag. by both radioimmunoassay and electron microscopy. There were 8 false positives by electron microscopy. It was concluded that there are particles in sedimented serum that are morphologically similar but unrelated to H.B. Ag. Therefore electron microscopy alone is not a reliable way to detect H.B. Ag. in serum. There was no evidence to suggest that H.B. Ag. is a cause of primary biliary cirrhosis.

SUMMARY 1. Thin-layer chromatography of porphyrin methyl esters provides a useful and rapid technique for resolving mixtures of porphyrins from biological samples. 2. Application of this technique to urinary, faecal and hepatic porphyrins from patients with symptomatic porphyria revealed five porphyrins of interest which could be identified by mass spectrometry as the methyl esters of porphyrins with 8-, 7-, 6-, 5- and 4-carboxyl groups. Millimolar extinction coefficients at the Soret absorption maxima for these porphyrin methyl esters in chloroform solution were measured. 3. A consistent pattern of urinary porphyrin excretion in symptomatic porphyria was seen with 8-, 7-, 6-, 5- and 4-carboxyl porphyrins constituting approximately 60%, 25%, 3%, 3% and 9% of the total respectively. Uroporphyrin (73%), heptacarboxylic porphyrin (26%) and hexa-carboxylic porphyrin (I %) were the only porphyrins detected in the liver of one patient. 4. Isomer analysis of excreted and hepatic porphyrins revealed that uroporphyrin was approximately 35% isomer I11 , hepta- and hexa-carboxylic porphyrins almost all isomer I11 and penta-carboxylic and coproporphyrin approximately 50% isomer 111. 5. To explain these findings it is suggested that there are two metabolic pathways for the handling of 6-aminolaevulic acid (ALA) in the liver. Symptomatic porphyria (porphyria cutanea tarda) is a well defined syndrome characterized clinically by increased mechanical fragility, blistering, pigmentation and superficial ulceration of the sun-exposed skin. A history of alcohol abuse and evidence of chronic parenchymatous liver disease can usually be elicited. The serum iron concentration is frequently raised and siderosis of the liver of varying degree is invariably present. Unlike inherited forms of hepatic porphyria, a positive family history of the disorder is exceptional and acute attacks are unknown. The disease is distinguished biochemically by a characteristic pattern of haem precursor